Small molecule modulation of protein-protein interactions: A. Microtubule-associated proteins affected by paclitaxel. B. Protein-protein interactions induced by bifunctional molecules.

摘要

No cellular protein acts alone. Protein interactions drive nearly every cellular process, from cell division, to metabolism, to programmed cell death. The structure and organization of cells is derived nearly entirely from protein-protein interactions. This dissertation explores two attempts to understand and control two different types of protein-protein interactions.; Part One explores the interactions of the anticancer drug Taxol ® (paclitaxel) with microtubules. Taxol binds to polymerized tubulin and prevents depolymerization, causing programmed cell death. The cellular events that occur between Taxol binding and programmed cell death are not known. Taxol association possibly triggers an unknown signal transduction event, by either attracting a protein to the microtubule surface or causing a protein to dissociate from the microtubule. This possibility was explored by screening MCF-7 cellular lysates against microtubule-based affinity chromatography columns. The screen identified proteins that interacted with microtubules in vitro with a decreased affinity in the presence of Taxol and other antimitotic small molecules. The key protein in this process appeared to be S12, a component of the regulatory unit of the 26S proteasome, which is involved in degrading key proteins during mitosis. The result suggested that S12 was localized to the microtubule, and that the presence of Taxol delocalized the protein and halted proper protein degradation, leading to programmed cell death. While the in vitro interaction of S12 with microtubules was further characterized, the association in vivo was not clearly established, and the Taxol dependence was not observed by other detection methods.; Part Two explores induced interactions between FKBP, involved in immunosuppression, and SH2 domains, involved in various signaling pathways. Previous experiments established that synthesized molecules could bind to both FKBP and SH2 domains, but the molecules would have an altered affinity for one protein if the other were present. In certain cases this interaction caused increased binding, in other cases decreased binding. The role of the linker between the two parts of the molecule was explored by the synthesis and testing of 17 bifunctional molecules with modified connectors. The linkers effected the distance and rotation of the molecule halves. The binding abilities of these bifunctional molecules were characterized through fluorescence polarization.